The present invention relates to new phenylserine derivatives as integrin antagonists with a broad spectrum of action having, inter alia, antiosteoporotic, antirestenotic, anticarcinogenic and antiatherosclerotic activity. The present invention moreover relates to the preparation of these compounds and their use for the production of medicaments, and also medicaments comprising them.
Integrins are heterodimeric transmembrane proteins found on the surface of cells, which play an important part in the adhesion of the cells to an extracellular matrix. They recognize extracellular glycoproteins such as fibronectin or vitronectin on the extracellular matrix by means of the RGD sequence occurring in these proteins (RGD is the single letter code for the amino acid sequence arginine-glycine-aspartate).
In general, integrins such as, for example, the vitronectin receptor, which is also called the xcex1vxcex23 receptor, or alternatively the xcex1vxcex25 receptor or the GpIIb/IIIa receptor play an important part in biological processes such as cell migration and cell-matrix adhesion and thus in diseases in which these processes are crucial steps. Cancer, osteoporosis, arteriosclerosis, restenosis (reoccurrence of stenosis after percutaneous transluminal angioplasty) and opthalmia may be mentioned by way of example.
The xcex1vxcex23 receptor occurs, for example, in large amounts on growing endothelial cells and makes possible their adhesion to an extracellular matrix. Thus the xcex1vxcex23 receptor plays an important part in angiogenesis, i.e. the formation of new blood vessels, which is a crucial prerequisite for tumor growth and metastasis formation in carcinoses. Furthermore, it is also responsible for the interaction between osteoclasts, i.e. cells resorbing mineralized tissue, and the bone structure. The first step in the degradation of bone tissue consists in the adhesion of osteoclasts to the bone. This cell-matrix interaction takes place via the xcex1vxcex23 receptor, which is why the corresponding integrin plays an important part in this process. Osteolytic diseases such as osteoporosis are induced by an inequilibrium between bone formation and bone destruction, i.e. the resorption of bone material caused by accumulation of osteoclasts predominates.
It was possible to show that the blockage of the abovementioned receptors is an important starting point for the treatment of disorders of this type. If the adhesion of growing endothelial cells to an extracellular matrix is suppressed by blocking their appropriate integrin receptors, for example, by a cyclic peptide or a monoclonal antibody, the endothelial cells die. Therefore angiogenesis does not occur, which leads to a cessation or resolution of the tumor growth (cf., for example, Brooks et al., Cell, Volume 79, 1157-1164, 1994).
Moreover, the invasive properties of tumor cells and thus their capability for metastasis formation are markedly decreased if their xcex1vxcex23 receptor is blocked by an antibody (Brooks et al., J. Clin. Invest., Volume 96, 1815, 1995).
The degradation of bone tissue can be suppressed by blockage of the xcex1vxcex23 receptors of the osteoclasts, since these are then unable to accumulate on the bone in order to absorb its substance (WO 98/18461, p. 1, 1. 24 to p. 2,1. 13).
By means of the blockage of the xcex1vxcex23 receptor on cells of the smooth aorta vascular musculature with the aid of integrin receptor antagonists, the migration of these cells into the neointima and thus angioplasty leading to arteriosclerosis and restenosis can be suppressed (Brown et al., Cardiovascular Res., Volume 28, 1815, 1994).
In recent years, compounds have therefore been sought which act as antagonists of integrin receptors. For example WO 97/24119 discloses victronectin (xcex1vxcex23 ) receptor antagonist as antiosteoporosis agents.
It was the object of the present invention to develop compounds which exhibit a high activity as integrin antagonists and in particular against the xcex1vxcex23 and/or the xcex1vxcex25 receptor.
The present invention relates to new compounds of the general formula (I) 
in which
R1 represents a radical of the formula 
in which
A represents a 5- to 7-membered saturated, partially unsaturated or aromatic heterocycle having up to three identical or different heteroatoms from the group consisting of N, O and/or S,
n denotes a number 0, 1, 2, 3 or 4,
R6 represents hydrogen, (C3-C8)-cycloalkyl, or straight-chain or branched (C1-C6)-alkyl, which for its part is optionally substituted by (C6-C10)-aryl, by 5- to 6-membered heteroaryl having up to three identical or different heteroatoms from the group consisting of N, O and/or S, or up to several times by halogen,
and
X denotes O, NH or a radical of the formula xe2x95x90Nxe2x80x94CN or xe2x95x90CHxe2x80x94NO2,
R2 and R3 are identical or different and denote hydrogen, halogen, (C1-C4)-alkyl or (C1-C4)-alkoxy,
R4 represents (C1-C6)-alkoxycarbonyl, (C6-C10)-arylmethyloxycarbonyl, (C1-C6)-alkylsulfonyl or (C3-C8)-cycloalkylsulfonyl,
or
represents 5- to 6-membered heteroarylsulfonyl having up to three identical or different heteroatoms from the group consisting of N, O and/or S, or (C6-C10)-arylsulfonyl, each of which is optionally substituted up to several times identically or differently by halogen, (C1-C4)-alkyl or (C1-C4)-alkoxy,
and
R5 denotes hydroxy, (C1-C6)-alkoxy or benzyloxy,
and their salts.
The compounds according to the invention can also be present in the form of their salts. In general, salts with organic or inorganic bases or acids may be mentioned here.
In the context of the present invention, physiologically acceptable salts are preferred. Physiologically acceptable salts of the compounds according to the invention can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Particularly preferred salts are, for example, those with hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
Physiologically acceptable salts can also be metal or ammonium salts of the compounds according to the invention. Those particularly preferred are, for example, sodium, potassium, magnesium or calcium salts, and also ammonium salts which are derived from ammonia, or organic amines, such as, for example, ethylamine, di- or triethylamine, ethanolamine, di- or triethanolamine, dicyclohexylamine, dimethyl-aminoethanol, arginine, lysine, ethylenediamine or 2-phenylethylamine.
The present invention also includes ammonium compounds which can be prepared by conversion of the free amines by means of alkylation.
In the context of the present invention, the substituents in general have the following meaning:
(C1-C6)-alkyl in general represents, depending on the abovementioned substituents, a straight-chain or branched hydrocarbon radical having 1 to 6, preferably 1 to 4 carbon atoms. Examples which may be mentioned are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl.
(C1-C6)-alkoxycarbonyl can be represented, for example, by the formula 
Alkyl here represents a straight-chain or branched hydrocarbon radical having 1 to 6 carbon atoms. Lower alkoxycarbonyl having 1 to 4 carbon atoms in the alkyl moiety is preferred. Examples which may be mentioned are the following alkoxycarbonyl radicals: methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert.-butoxycarbonyl or isobutoxycarbonyl.
(C3-C8)-cycloalkyl in general represents a cyclic hydrocarbon radical having 3 to 8 carbon atoms. Cyclopropyl, cyclopentyl and cyclohexyl are preferred. Examples which may be mentioned are cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
(C6-C10)-aryl in general represents an aromatic radical having 6 to 10 carbon atoms. Preferred aryl radicals are phenyl and naphthyl.
Halogen in the context of the invention represents fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.
(C1-C6)-alkoxy in general represents, depending on the abovementioned substituents, a straight-chain or branched hydrocarbon radical bonded via an oxygen atom and having 1 to 6 carbon atoms. Lower alkoxy having 1 to 4 carbon atoms is preferred. An alkoxy radical having 1 to 3 carbon atoms is particularly preferred. Examples which may be mentioned are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, isopentoxy, hexoxy, isohexoxy.
(C1-C6)-alkylsulfonyl can be represented by the formula 
wherein alkyl represents a straight-chain or branched hydrocarbon radical having 1 to 6, preferably 1 to 4 carbon atoms. Examples which may be mentioned are: methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl.
(C6-C10)-arylsulfonyl can be represented by the formula 
wherein aryl is a (C6-C10)-aryl as defined herein.
Heteroarylsulfonyl can be represented by the formula 
wherein Het represents a 5- or 6-membered heterocycle as defined below.
5- or 7-membered heterocycles in the context of the invention, depending on the abovementioned substituents, in general represent a 5- or 7-membered, preferably 5- to 6-membered saturated, unsaturated or aromatic heterocycle which can contain up to 3 heteroatoms selected from S, N and O and which can optionally also be bound via a nitrogen atom. Examples which may be mentioned are: pyridyl, pyrimidinyl, oxazolyl, thienyl, furyl, pyrrolyl.
The compounds according to the invention can exist in stereoisomeric forms which either behave as image and mirror image (enantiomers), or which do not behave as image and mirror image (diastereomers). The invention relates both to the enantiomers or diastereomers and their respective mixtures. These mixtures of the enantiomers and diastereomers can be separated into the stereoisomerically uniform constituents in a known manner.
Preferred compounds of the general formula (I) according to the invention are those in which
R1 represents a radical of the formula 
in which
A represents a 5- to 6-membered aromatic heterocycle having up to three identical of different heteroatoms from the group consisting of N, O and/or S,
n denotes the number 1 or 2,
and
R6 represents hydrogen, (C3-C5)-cycloalkyl, (C6-C10)-arylmethyl, 5- to 6-membered heteroarylmethyl having up to three identical of different heteroatoms from the group consisting of N, O and/or S, or straight-chain or branched (C1-C4)-alkyl, which is optionally substituted up to three times by fluorine or chlorine,
R2 and R3 are identical or different and denote hydrogen, fluorine, methyl or methoxy,
R4 represents (C1-C4)-alkoxycarbonyl, benzyloxycarbonyl, (C1-C4)-alkylsulfonyl or (C3-C5)-cycloalkylsulfonyl,
or
represents 5-membered heteroarylsulfonyl having up to three identical or different heteroatoms from the group consisting of N, O and/or S, or phenylsulfonyl, each of which is optionally substituted up to three times identically or differently by fluorine, chlorine, methyl or ethyl,
and
R5 denotes hydroxy, methoxy, ethoxy or benzyloxy,
and their salts.
Particularly preferred compounds of the general formula (I) according to the invention are those in which
R1 represents a radical of the formula 
in which
R6 represents cyclopropyl, cyclobutyl, benzyl, pyridylmethyl, or straight-chain or branched (C1-C3)-alkyl, which is optionally substituted up to three times by fluorine,
R2 and R3 each denote hydrogen,
R4 represents phenylsulfonyl or 1,2- or 1,3-oxazolylsulfonyl, each of which is optionally substituted up to three times identically or differently by fluorine, chlorine or methyl,
and
R5 denotes hydroxy, methoxy or ethoxy,
and their salts.
Furthermore, a process for the preparation of the compounds of formula (I) was found wherein a compound of formula (II) 
wherein
R3 has the meaning indicated above,
R5xe2x80x2 has the meaning of R5 as indicated above, except hydroxy,
Tr represents the triphenyl methane group, and
E represents halogen, preferably bromine or iodine, is coupled with a compound of formula (III) 
wherein R1xe2x80x2 represents amino, nitro or formyl,
R2 has the meaning indicated above, and
G represents a tri-(C1-C4)-alkylstannyl or a di-hydroxy or di-(C1-C4)-alkoxy boron group, preferably xe2x80x94B(OH)2 in an inert solvent in the presence of a catalyst, to yield compounds of formula (IV) 
wherein R1xe2x80x2, R2, R5xe2x80x2 and Tr have the meaning indicated above and from which Tr is cleaved off and the resulting amino group is reacted with a reagent
R4xe2x80x94Y 
wherein Y represents a leaving group, preferably chlorine, and R4 has the meaning indicated above,
and finally, using standard methods, R1xe2x80x2 and R5xe2x80x2 are converted into the desired substituents as necessary.
The coupling reaction is preferably a Suzuki or Stille reaction.
Suitable catalysts are palladium compounds such as Pd(PPh3)2Cl2.
The coupling reaction should preferably be run under mild conditions, i.e. at temperatures from 0xc2x0 to 50xc2x0 C., preferably at room temperature, to avoid undesired side reactions. It has been found that good yields under mild conditions can be obtained if the ligand triphenylphosphine is exchanged by addition of other ligands, preferably AsPh3.
Furthermore, the coupling reaction may be carried out in the presence of a base. Suitable bases are for example alkaline and alkaline earth metal carbonates, bicarbonates, fluorides, tertiary organic amines, preferably Cs2CO3.
The compounds of formula (II) can be obtained by condensation reaction of compounds of formula (V) 
wherein
R3 and E have the meaning indicated above
with compounds of formula (VI) 
wherein R5xe2x80x2 and Tr have the meaning indicated above.
For the condensation reaction mild reaction conditions are preferred, such as temperatures from 0xc2x0 C. to 50xc2x0 C., preferably about room temperature.
It is advantageous to use a suitable condensation agent, preferably standard Mitsunobu reaction conditions, such as the system diethyl azodicarboxylate (DEAD)/triphenylphosphine (PPh3).
The solvent used should be inert under the reaction conditions, such as for example toluene.
A preferred embodiment of the complete synthesis is exemplified in the following representative synthetic scheme: 
Representative Synthetic Scheme
The compounds of the formula (I) according to the invention have a surprisingly wide spectrum of pharmacological action.
The compounds according to the invention exhibit an antagonistic action against integrin inhibitors, in particular the xcex1vxcex23 receptor or the xcex1vxcex25 receptor.
They can be used as active compounds in medicaments for the reduction of changes to vascular walls and are employed for the treatment of arterial hypertension and atherosclerosis. Moreover, they can be employed for the treatment of coronary heart disorders, cardiac insufficiency, disorders of brain function, ischemic brain disorders, (peripheral) circulation disorders, microcirculation disorders and thromboses, functional disorders of the kidney and adrenal gland, bronchospastic and vascular system-related disorders of the airways, sodium retention and edemas as well as osteolytic disorders such as osteoporosis, cancer, carcinoses and ophthalmic diseases.
Furthermore, the proliferation and migration of smooth muscle cells plays a decisive part in the occlusion of vessels. The compounds according to the invention are suitable for inhibiting this proliferation and can therefore also be employed for the treatment of restenosis.
The novel active compounds are distinguished pharmacologically by good kinetic parameters. In particular, they have favorable properties with respect to clearance.
The novel active compounds can be converted in a known manner into the customary formulations, such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, nontoxic, pharmaceutically suitable excipients or solvents. In this case the therapeutically active compound should in each case be present in a concentration of approximately 0.5 to 90% by weight of the total mixture, i.e. in amounts which are sufficient in order to achieve the dosage range indicated.
The formulations are prepared, for example, by extending the active compounds with solvents and/or excipients, if appropriate using emulsifiers and/or dispersants, where, for example, if water is used as a diluent organic solvents can optionally be used as auxiliary solvents.
Administration is carried out in a customary manner, preferably orally or parenterally, in particular perlingually or intravenously.
In the case of parenteral administration, solutions of the active compound using suitable liquid carrier materials can be employed.
In general, it has proven advantageous in the case of intravenous administration to administer amounts from approximately 0.001 to 1 mg/kg, preferably approximately 0.01 to 0.5 mg/kg, of body weight to achieve effective results, and in the case of oral administration the dose is approximately 0.01 to 100 mg/kg, preferably 0.1 to 50 mg/kg, of body weight.
In spite of this, if appropriate, it may be necessary to depart from the amounts mentioned, namely depending on the body weight or the type of application route, on individual behavior toward the medicament, the manner of its formulation and the time or interval at which administration takes place. Thus in some cases it may be adequate to manage with less than the abovementioned minimum amount, while in other cases the upper limits mentioned must be exceeded. In the case of the administration of relatively large amounts, it may be advisable to divide these into several individual doses over the course of the day.
xcex1vxcex23 inhibitory activity
The substances were tested for their ability to inhibit xcex1vxcex23/echistatin binding analogously to Kumar C. C., Nie H. M., Rogers G. P., Malkowski M., Maxwell E., Catino J. J. and Armstrong L. (Journal of Pharmacology and Experimental Therapeutics 283(2)(1997)843-853).
xcex1vxcex23 from human placenta (Smith J. W. and Cheresh, D. A. (1988), J. Biol. Chem. 263, 18726-18732) (1 mg/ml 50 mM tris HCl pH 7.4, 100 mM NaCl, 2 mM CaCl2, 1% and octylglucoside) was diluted with test buffer (50 mM tris-HCl pH 7.4, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1% bovine serum albumin) and 55 xcexcl each of this xcex1vxcex23 solution were added to the wells of a 96-well microtiter plate (about 0.1-0.3 xcexcg of xcex1vxcex23 per well). 2 xcexcl of the substances to be tested dissolved in DMSO were then added. 10 xcexcl (40,000 cpm) of I125-echistatin per well were then added and the mixture was incubated for 1 hour at room temperature with careful shaking. It was then treated with 100 xcexcg of wheatgerm-coated yttrium silicate beads (Amersham, type RPNQ0011) in 25 xcexcl of distilled water. After 1 hour at room temperature, the cpm values were measured in a scintillation counter. The Ki values were determined in duplicate from concentration series. The nonspecific binding was determined in the presence of 0.1 xcexcM unlabeled echistatin or by addition of 5 mM EDTA to the binding mixture.
Results:
The present invention is illustrated in greater detail below by working examples.
Experimental Part